Brief background on my blood samples storage: A portion of the blood samples (24) were traceable to individual animals National Livestock Identification System ear tag, these were inverted 10 times to throughly mix the blood prior to freezing to ensure the sample was frozen as whole blood; while a portion of the remaining samples that weren't traceable were allowed to remain separated into its plasma and haemoglobin constituteunts (hope thats the right tech terms) and frozen.

All the samples were taken from the freezer last week and placed into a refrigerator at 4 d C to thaw slowly overnight. The next day I did a practice run on two blood samples (link below), which worked fine i.e. DNA precipitated into the FG3 buffer. One sample was blood that was mixed before freezing at -20 d C and mixed after thawing. While the other sample had separated into the plasma at top before freezing and I have left these samples unmixed, as I decided to experiment with 3 individual samples (i.e. # 2, 3, 4 as shown in linked photos) to see if I could extract DNA from the plasma (#2), the middle layer (#3 i.e. bottom mix of plasma and top layer of blood combined) and a sample from the base of the vial (#4).

See my photos (excludes image of large EDTA vial containing mixed whole blood, which just looks like normal)

Anyway. I've got to the end of the protocol included with the kit, using the one on page 14 based on 500 microlitres (ul) of whole blood. Instead of isopropanol, I used ethanol (100%) and doubled the rate to 300ul.

The practice run using the 4 test samples went fine without any problems. However when I ran the actual samples none of the 24 DNA samples precipitated into the buffer.

I've added extra FG3 buffer, the protocol lists 200ul FG3, which I increased to 300ul and found this to be insufficient. I incubated for a further hour at 65 d C in a water bath, with no success except for one sample that still had DNA particles visible to the naked eye.

Then I incubated overnight at 55 d C for 20 hours and two more samples were partially dissolved with small particles still visible to naked eye.

I placed the samples in a device with a rotating platform that heats to 60 d C and left for an hour, though no change occurred.

I tried to vortex several samples at the maximum vortex speed to break up the DNA by sheer force and some of the samples containing lighter DNA partially dissolved though not completely and the darker DNA samples were practically unbreakable. Given one sample was being vortexed for around 2 mins and the supernatant (FG3 buffer) was able to penetrate between the lid and force its way out of the microfuge tube, I figured a contamination issue may entail and had to change my gloves between samples, so I gave up on this idea. If I didn't hold the tube sideways, I could control these issue, though it meant the process would take much longer. Time is not on my side.

Next I put the samples into a heat incubator oven for an hour at 60 d C. This didn't work either, the DNA was still present and just as tough to dissolve.

Then I vortexed all the samples for 15 mins at 17,000 x g 15 d C, which allowed the DNA to stick to the bottom of the microfuge tube, though requires careful handling not to dislodge as I wanted to discard some of the supernatant from a couple of samples for further experimental testing. See below.

Following I took 8 samples and split them into two groups (see image of the hand written table and notes), note: these samples are not shown in the images linked to this discussion. In group 1, an additional volume of FG 3 buffer was added, this meant two samples had 400ul and the other two had 500ul. Out of the group 1 microfuge tube samples I labelled one 400ul sample 'V' (for vortex) and one 500ul sample 'V', the other two I will as non-vortex. This is clearly demonstrated in the image of the table (left side) I hand wrote as a visual aide.

The samples in group 2 on the otherhand, I decided to discard the FG 3 buffer supernatant and replace with fresh FG 3 buffer, see image of hand written table (right side) titled 'FG# replaced with fresh FG3 (total)', again two samples were vortexed as they were for group 1.

Interestingly enough, the group 2 fresh FG 3 buffer replacement together with vortexing did break up the dna and it was largely dissolved, though still minor particles visible to naked eye. The group 1 samples that I vortex, did seem to break up DNA better when the volume of additional FG3 was 500 ul, though it took longer to break up and more effort on the vortex than group 2 samples which were vortexed.

I placed these eight samples in the incubator heat oven at 60 d C (hottest temp.), which at present has been 1 hour and 30 mins. I am going to vortex both group 1 and 2 then go from there.

Any comments, feedback, suggestions or references please let me know.

Regards,

Jess Hughes

-agorganic-

agorganic on Tue Sep 4 05:29:54 2012 said:

The practice run using the 4 test samples went fine without any problems. However when I ran the actual samples none of the 24 DNA samples precipitated into the buffer.

I've added extra FG3 buffer, the protocol lists 200ul FG3, which I increased to 300ul and found this to be insufficient. I incubated for a further hour at 65 d C in a water bath, with no success except for one sample that still had DNA particles visible to the naked eye.

Then I incubated overnight at 55 d C for 20 hours and two more samples were partially dissolved with small particles still visible to naked eye.

I tried to vortex several samples at the maximum vortex speed to break up the DNA by sheer force and some of the samples containing lighter DNA partially dissolved though not completely and the darker DNA samples were practically unbreakable. Given one sample was being vortexed for around 2 mins and the supernatant (FG3 buffer) was able to penetrate between the lid and force its way out of the microfuge tube, I figured a contamination issue may entail and had to change my gloves between samples, so I gave up on this idea. If I didn't hold the tube sideways, I could control these issue, though it meant the process would take much longer. Time is not on my side.

Next I put the samples into a heat incubator oven for an hour at 60 d C. This didn't work either, the DNA was still present and just as tough to dissolve.

Then I vortexed all the samples for 15 mins at 17,000 x g 15 d C, which allowed the DNA to stick to the bottom of the microfuge tube, though requires careful handling not to dislodge as I wanted to discard some of the supernatant from a couple of samples for further experimental testing. See below.

Interestingly enough, the group 2 fresh FG 3 buffer replacement together with vortexing did break up the dna and it was largely dissolved, though still minor particles visible to naked eye. The group 1 samples that I vortex, did seem to break up DNA better when the volume of additional FG3 was 500 ul, though it took longer to break up and more effort on the vortex than group 2 samples which were vortexed.

It sounds like you have either left your samples to dry for too long after the DNA has been isolated, this can make it very hard to dissolve; or you have precipitated a lot of protein with the DNA, which also is hard to dissolve and will be a whiteish pellet rather than than the clear/transparent pellet, that you would expect for DNA. It is quite important that a minimum of the protein from the procedure come through, so ensure that the digestion reaction has fully taken place.

What you should do is run a small amount of the DNA on a 0.8% agarose gel and also quantitate with a spectrophotometer (a nanodrop or similar device is good if you have one available). These will tell you two things - one, if you have managed to isolate DNA; and two, how pure it is.

YOu may have also confused a bit of terminology - vortexing is a method of mixing and usually (never) comes with a g force measurement. Centrifugation is the thing that pellets the DNA and is measured in g forces (actually relative centrifugal forces or RCF if you want the proper terminology). Precipitation is also where things come out of solution, the opposite of dissolving.

-bob1-

Hi bob, thankyou for reply. That was a typo, should have been centrifuge. I didnt know about the protein. The vortexing seems to merely disintegrate the DNA, though perhaps I can perform your suggestion and assess the DNA quality.

-agorganic-

The kit instructions that I found on the web suggest that there is a protease step (step 4) in the protocol - this should digest the protein fairly effectively such that you shouldn't get much coming through. However, proteases are not particularly stable, so if it has either been stored improperly, or not added to buffer FG2 before this step, then it won't work. You also need to ensure that the pellet from step 3 has been fully resuspended at this point - any lumps will cause protein carry over into the DNA precipitation step.

Make sure that you are using the volumes of blood recommended in the protocol - too much will cause there to be too much protein and possibly other things in there that will hinder you from getting (relatively) clean DNA, and may also inhibit downstream applications like PCR or restriction digest.

If you want really clean DNA, you can further purify by doing either a Phenol:chloroform or a chloroform extraction.

-bob1-

Hi Thelomitra,

Thanks for the reply. I should have added a link to the kit instruction. I realised there was a problem with my test procedure where I made a mistake by increasing the volume of blood used (500ul) though followed the protocol using reagent volume for only 300ul of blood, therefore the protease/FG2 buffer was sufficient although insufficient volume of FG1, ethanol (70% and 100% - substituted for isoproponol) and FG3 (which I was did increase as it was the last step).

I also had been vortexing the microfuge tubes at maximum speed for several minutes in an attempt to speed up the break down of DNA, I was informed by someone from my uni this morning that vortexing might be shearing the DNA and that it is quite fragile.

On friday I had restarted the tests again making sure to follow the protocol for the lowest volume of blood (100ul) and associated reagent volumes, again I substituted ethanol (100%) for isoproponol and upon reaching step 13 in a water bath I viewed the samples flicking several samples gently and noticed a couple of samples DNA dissolved into the FG3 supernatant. However several other samples DNA didn't dissolve and for experimentation purposes, I vortexed a couple of samples, now its difficult to say what dissolved and what just disintegrated leaving behind small particles that were visible to the naked eye. The only thing which I shouldn't have done was centrifuge for 4 mins at step 7, rather than only 3 mins.

I concur if I undertake a 3rd test, I will refrain from vortexing the dna and instead incubate in the water bath overnight as the protocol describes in the very last NOTE. The completed second test samples which were particularly dense and hadn't dissolved are now back in the water bath incubating at 65 d C, which I will leave overnight and assess the results tomorrow.

The lady from uni that I spoke to today, mentioned that I could just add ethanol to the FG3, though she wasn't sure what volume of ethanol to add and thought there was some kind of ratio involved depending on the volume of FG3 buffer. Unfortunately she couldn't recall what sort of procedure to follow, do you have any ideas?

Regards,

Jess Hughes

-agorganic-

agorganic on Mon Sep 10 02:51:55 2012 said:

I also had been vortexing the microfuge tubes at maximum speed for several minutes in an attempt to speed up the break down of DNA, I was informed by someone from my uni this morning that vortexing might be shearing the DNA and that it is quite fragile.

This is true, but is relatively unlikely - I have never had a problem with vortexing DNA. In fact, some protocols call for vortexing of the DNA to re-suspend it.

agorganic on Mon Sep 10 02:51:55 2012 said:

On friday I had restarted the tests again making sure to follow the protocol for the lowest volume of blood (100ul) and associated reagent volumes, again I substituted ethanol (100%) for isoproponol and upon reaching step 13 in a water bath I viewed the samples flicking several samples gently and noticed a couple of samples DNA dissolved into the FG3 supernatant. However several other samples DNA didn't dissolve and for experimentation purposes, I vortexed a couple of samples, now its difficult to say what dissolved and what just disintegrated leaving behind small particles that were visible to the naked eye. The only thing which I shouldn't have done was centrifuge for 4 mins at step 7, rather than only 3 mins.

This all should be fine. Longer centrifugation should pellet more DNA and the protocol recommends it if the pellets are loose.

agorganic on Mon Sep 10 02:51:55 2012 said:

The lady from uni that I spoke to today, mentioned that I could just add ethanol to the FG3, though she wasn't sure what volume of ethanol to add and thought there was some kind of ratio involved depending on the volume of FG3 buffer. Unfortunately she couldn't recall what sort of procedure to follow, do you have any ideas?

I'm not quite sure why you would want to add EtOH to the FG3, this would prevent your DNA from dissolving. If you wanted to re-precipitate the DNA, you could add some more salt and then 2-2.5 x volume of 100% EtOH.

I didn't see how you were doing it in your original post but did you make the FG2/protease mix fresh each time, and did the colour change from red to olive green during step 5?

-bob1-

bob1 on Mon Sep 10 05:11:09 2012 said:

I didn't see how you were doing it in your original post but did you make the FG2/protease mix fresh each time, and did the colour change from red to olive green during step 5?

Thanks for your help and answers Bob1. The protease was resuspended with 300ul of FG3 i.e. before the 4 (sample) practice tests and used the remaining portion of FG3 resuspended protease for both test 1 and test 2 also, which I had accidentally left it out on the bench for an hour or so before test 1 at room temperature and returned to 4 d C as soon as I realised. At step 4, yes I did make the FG2/(resuspended) qiagen protease fresh each time and used within an hour of producing.

During the first test that I realised I missed up, the protease/FG2 buffer would have incorrectly been for 500ul (rather than 300ul) of blood based on 24 samples, where I used the following calculation to create the FG2/protease:

24 whole blood samples * 0.5ml = 12ml whole blood (total)

=> FG2 total = 12 * 0.5ml
= 6 ml

plus protease = 12 * 0.005
= 0.060ml
= 60ul

Although I had not increased the reagents volumes for 500ul of blood in this '1st' test, instead mistakenly followed the standard protocol as listed in the instructions that is set out for only 300ul of blood.

The 2nd (new) repeat test I decided to follow column 1 shown in table 2 (p. 14) of the flexigene kit protocol which uses 100ul of blood, 250 FG1 (@ step 1), 50ul FG2/protease mixture (@ step 4), 100ul of 100% ethanol (@ step 6) which I substituted for isoproponol, 50ul of 70% ethanol (@ step 9) and 100ul of FG3 (@ step 13); this last stepped I even increased the FG3 volume to 500ul total in each microfuge tube, which seemed to help dissolve the DNA in some samples, with most of the samples needing vortexing to disintegrate the DNA. As part of this 2nd test, I followed column 1 of table 3 (p. 15) with 50ul of FG2 and 0.5ul of

I'm not using specialised pippette tips, so there is some retention of blood (5-10ul blood??) remaining on the sides of the discarded pippette tips and as they warm to room temperature, though the fresh pippette tips don't retain any significant volume of the reagents...Perhaps the slightly reduced volume of blood is not important. I'm just trying to think out of the box.

In my last post I was going to leave the samples in the water bath over night, though instead I took out after one hour and then placed into a heat incubator that has a rotating pad which I set for 999 mins (the maximum) and will assess again tomorrow.

To one of the samples with dense (in appearance), stubborn DNA I added 1.5 ml total of FG3 buffer, it perhaps helped to soften and break apart the DNA pellet so it wasn't so dense. I'm not willing to replicate this with other samples in case my FG3 buffer runs out and I need to run test 3, so difficult to know how effective that was at the moment!! lol. I'm back home and will go back again to uni in the morning to re-evaluate the situation, come what may!

Hopefull this makes sense.

Regards,

Jesse

-agorganic-

Yes samples did change from red to olive

-agorganic-

agorganic on Mon Sep 10 06:34:49 2012 said:

The 2nd (new) repeat test I decided to follow column 1 shown in table 2 (p. 14) of the flexigene kit protocol which uses 100ul of blood, 250 FG1 (@ step 1), 50ul FG2/protease mixture (@ step 4), 100ul of 100% ethanol (@ step 6) which I substituted for isoproponol, 50ul of 70% ethanol (@ step 9) and 100ul of FG3 (@ step 13); this last stepped I even increased the FG3 volume to 500ul total in each microfuge tube, which seemed to help dissolve the DNA in some samples, with most of the samples needing vortexing to disintegrate the DNA. As part of this 2nd test, I followed column 1 of table 3 (p. 15) with 50ul of FG2 and 0.5ul of

this looks fine, as do your calculations. It shouldn't really matter, but it may be best to try it with the isopropanol if you can, in case they are using some special salt or something that enhances precipitation with isopropanol.

agorganic on Mon Sep 10 06:34:49 2012 said:

I'm not using specialised pippette tips, so there is some retention of blood (5-10ul blood??) remaining on the sides of the discarded pippette tips and as they warm to room temperature, though the fresh pippette tips don't retain any significant volume of the reagents...Perhaps the slightly reduced volume of blood is not important. I'm just trying to think out of the box.

This definitely shouldn't be a problem either, so long as the ratios are roughly the same in the reactions, they should still work fine.

agorganic on Mon Sep 10 06:34:49 2012 said:

To one of the samples with dense (in appearance), stubborn DNA I added 1.5 ml total of FG3 buffer, it perhaps helped to soften and break apart the DNA pellet so it wasn't so dense. I'm not willing to replicate this with other samples in case my FG3 buffer runs out and I need to run test 3, so difficult to know how effective that was at the moment!! lol. I'm back home and will go back again to uni in the morning to re-evaluate the situation, come what may!

I can't see where you are going wrong at all, but not having used this kit, or even extracted DNA from blood using a kit (I did it the old fashioned way, buffy coats and all), there may be something I am missing. The only things I can come up with are that either you are drying the DNA for too long, which makes it more or less insoluble in the short term, or that there is a lot of protein coming through the preparation, and this is what you are seeing. However, over-dried DNA should still dissovle with a bit of heating and time, and the indicators are that your protease is working. You could try adding some other protease and see if that makes a difference.

Some other things you could try: Get someone experienced in using the kit to have a watch while you work - there may be something simple that you are or aren't doing that may make all the difference.
Try extracting the DNA in a different manner, perhaps with a different kit.
Ring up Qiagen and/or check out their technical help on the web and see what their advice is.

-bob1-

Thanks bob.

I am going to run test 3 tomorrow but double the concentration of protease/fg2 buffer (from 0.5ul/50ul to 1ul/50ul). i will look at obtaining some isoproponol to follow protocol as u mentioned and started also looking at the uv absorbance of samples with spectrometer of test 1, test 2 and test 3 (when it's complete) just to cross reference techniques.